DNA damage is the first step in the initiation of cancer. Every human cell experiences approximately 50,000 base damage/removal events and ~1000-2000 base mispair errors per day, Since DNA alterations vary from one DNA molecule to another it is important to detect such lesions in individual molecules. The primary objective of this R21 proposal is to develop a methodology for detecting DNA lesions at the single molecule level using Atomic Force Microscopy (AFM) techniques. Lesions will be detected either directly, by force spectroscopy and/or by imaging with carbon nanotube (SWNT) AFM tips, or indirectly, by detecting the secondary changes induced in DNA by the repair proteins. These secondary changes include e.g. strand incision, unwinding, degradation and re-synthesis. They are co-localized with the primary lesion and should be easily detected by our AFM assay. To achieve our objective we need to optimize the AFM platform to achieve: Angstrom level precision in the x, y, and z-axis and force errors below 10 pN. The platform must be able to align a molecule with the pulling direction and an ability to set a limit on the pulling force in order to preserve the coupling of the molecule to the instrument. We will construct various DNA molecules with model lesions and use AFM imaging and force spectroscopy to determine their fingerprints. We will study the effect of UV radiation on DNA elasticity and the course of its photoreactivation. A second objective is to explore the use of AFM spectroscopy and imaging to study the mismatch repair (MMR) in E. coil and in humans. We will determine force spectrograms of DNA molecules containing a mismatched base in the presence of bacterial and human repair activities. We will record the changes of DNA elasticity during the course of repair. We will also visualize primary lesions, repair-induced secondary alterations and DNA/proteins complexes. These measurements will allow us to examine key intermediates in the repair reaction and to further clarify molecular functions of the repair proteins and their assemblies. The proposed experiments will use for the first time, AFM-based single-molecule force spectroscopy to detect DNA lesions and to examine the course of a repair reaction. The findings should be of great significance for nanoscale DNA diagnostics and DNA damage and repair research.